Lignin-carbohydrate complexes from sisal (Agave sisalana) and abaca (Musa textilis): chemical composition and structural modifications during the isolation process.

MAIN CONCLUSION: Two types of lignins occurred in different lignin-carbohydrate fractions, a lignin enriched in syringyl units, less condensed, preferentially associated with xylans, and a lignin with more guaiacyl units, more condensed, associated with glucans. Lignin-carbohydrate complexes (LCC) were isolated from the fibers of sisal (Agave sisalana) and abaca (Musa textilis) according to a plant biomass fractionation procedure recently developed and which was termed as "universally" applicable to any type of lignocellulosic material. Two LCC fractions, namely glucan-lignin (GL) and xylan-lignin (XL), were isolated and differed in the content and composition of carbohydrates and lignin. In both cases, GL fractions were enriched in glucans and comparatively depleted in lignin, whereas XL fractions were depleted in glucans, but enriched in xylans and lignin. Analysis by two-dimensional Nuclear Magnetic Resonance (2D-NMR) and Derivatization Followed by Reductive Cleavage (DFRC) indicated that the XL fractions were enriched in syringyl (S)-lignin units and ß-O-4' alkyl-aryl ether linkages, whereas GL fractions have more guaiacyl (G)-lignin units and less ß-O-4' alkyl-aryl ether linkages per lignin unit. The data suggest that the structural characteristics of the lignin polymers are not homogeneously distributed within the same plant and that two different lignin polymers with different composition and structure might be present. The analyses also suggested that acetates from hemicelluloses and the acyl groups (acetates and p-coumarates) attached to the γ-OH of the lignin side chains were extensively hydrolyzed and removed during the LCC fractionation process. Therefore, caution must be paid when using this fractionation approach for the structural characterization of plants with acylated hemicelluloses and lignins. Finally, several chemical linkages (phenylglycosides and benzyl ethers) could be observed to occur between lignin and xylans in these plants.

Obtaining biobleached eucalyptus cellulose fibres by using various enzyme combinations.

Various combinations of laccases, xylanase and cellulase were used to biobleach cellulose fibres from eucalyptus. The Trametes villosa and Myceliophthora thermophila laccases were used in combination with violuric acid (VA(TvL) system) and methyl syringate (MeS(MtL) system), respectively, as mediator. A dissimilar mode of action of the two systems was found: the VA(TvL) treatment released both hexenuronic acids and lignin, whereas the MeS(MtL) released lignin alone. Pulp properties were further improved by applying the mediator before the enzyme during treatment. Pulp properties comparable to those provided by industrial TCF sequences were obtained by inserting a xylanase pretreatment before VA(TvL), but no significant effect was observed after the cellulase pretreatment. As an added value, the resulting enzymatically bleached fibres possess a reduced hexenuronic acid content. The chemical oxygen demand of the effluents from each stage was also assessed.

Demonstration of laccase-based removal of lignin from wood and non-wood plant feedstocks.

The ability of Trametes villosa laccase, in conjuction with 1-hydroxybenzotriazole (HBT) as mediator and alkaline extraction, to remove lignin was demonstrated during treatment of wood (Eucalyptus globulus) and non-wood (Pennisetum purpureum) feedstocks. At 50 Ug(-1) laccase and 2.5% HBT concentration, 48% and 32% of the Eucalyptus and Pennisetum lignin were removed, respectively. Two-dimensional nuclear magnetic resonance of the feedstocks, swollen in dimethylsulfoxide-d(6), revealed the removal of p-hydroxyphenyl, guaiacyl and syringyl lignin units and aliphatic (mainly ß-O-4'-linked) side-chains of lignin, and a moderate removal of p-coumaric acid (present in Pennisetum) without a substantial change in polysaccharide cross-signals. The enzymatic pretreatment (at 25 Ug(-1)) of Eucalyptus and Pennisetum feedstocks increased the glucose (by 61% and 12% in 72 h) and ethanol (by 4 and 2 g L(-1) in 17 h) yields from both lignocellulosic materials, respectively, as compared to those without enzyme treatment.

On hexenuronic acid (HexA) removal and mediator coupling to pulp fiber in the laccase/mediator treatment.

Flax soda/AQ pulps were treated with different fungal laccase-mediator combinations followed by physical and chemical characterization of the pulps to obtain a thorough understanding of the laccase/mediator effects on hexenuronic acid (HexA) removal and the coupling of mediator onto pulps for fiber functionalization. Large differences were found and the presence of lauryl gallate (LG) during Trametes villosa laccase (TvL) treatment (TvL+LG) resulted in a much larger reduction of pulp-linked HexA than the combination of p-coumaric acid (PCA) and Pycnoporus cinnabarinus laccase (PcL). A major portion of LG became attached to the pulp as revealed by an increase in the kappa number and further confirmed by thioacidolysis and (1)H NMR analysis of solubilized pulp fractions. Additional experiments with other chemical pulps and isolated pulp xylan and lignin revealed that HexA seems to be the sole pulp component attacked by TvL+LG. As a substrate for TvL, the reaction preference order is PCA>HexA>LG.

Use of cellulases and recombinant cellulose binding domains for refining TCF kraft pulp.

The modular endoglucanase Cel9B from Paenibacillus barcinonensis is a highly efficient biocatalyst, which expedites pulp refining and reduces the associated energy costs as a result. In this work, we set out to identify the specific structural domain or domains responsible for the action of this enzyme on cellulose fibre surfaces with a view to facilitating the development of new cellulases for optimum biorefining. Using the recombinant enzymes GH9-CBD3c, Fn3-CBD3b, and CBD3b, which are truncated forms of Cel9B, allowed us to assess the individual effects of the catalytic, cellulose binding, and fibronectin-like domains of the enzyme on the refining of TCF kraft pulp from Eucalyptus globulus. Based on the physico-mechanical properties obtained, the truncated form containing the catalytic domain (GH9-CBD3c) has a strong effect on fibre morphology. Comparing its effect with that of the whole cellulase (Cel9B) revealed that the truncated enzyme contributes to increasing paper strength through improved tensile strength and burst strength and also that the truncated form is more effective than the whole enzyme in improving tear resistance. Therefore, the catalytic domain of Cel9B has biorefining action on pulp. Although cellulose binding domains (CBDs) are less efficient toward pulp refining, evidence obtained in this work suggests that CBD3b alters fibre surfaces and influences paper properties as a result.

Can the laccase mediator system affect the chemical and refining properties of the eucalyptus pulp?

Application of a laccase mediator system (an L stage) to TCF and ECF bleached pulp from Eucalyptus globulus with low residual lignin content (KN(lig)1.0) provides useful information about its effects on hexenuronic acids, functional groups (carboxyl and carbonyl) and electrokinetic properties such as zeta potential and surface charge. The use of laccase from Trametes villosa in combination with the mediator 1-hydroxybenzotriazole (HBT) was found to oxidize cellulose to carbonyl groups and reduce the amount of carboxyl groups present in TCF pulp by effect of its partially removing hexenuronic acids from it. This result may open up new prospects for improving brightness stability in pulp. In addition, the laccase mediator system modifies the surface charge and zeta potential in the fibre suspension for the removal of ionizables groups in TCF pulp. This result has no adverse effect on the pulp refining efficiency. L treatment requires less mechanical energy than conventionally refined pulp to obtain an optimal tensile and tear index in handsheets. This behavior may be attributable to the modification of the electrokinetic properties.

Influence of the hexenuronic acid content on refining and ageing in eucalyptus TCF pulp.

Brightness in totally chlorine-free (TCF) pulp is more unstable than it is in elemental chlorine-free (ECF) pulp, seemingly by effect of the former containing greater amounts of oxidizable structures such as hexenuronic acids (HexA). Accelerated ageing tests involving the application of moist heat to pulp revealed that brightness reversion can be alleviated by using an effective biotechnological method involving an enzyme-mediator treatment. Thus, the joint use of laccase from Trametes villosa and the mediator hydroxybenzotrialoze (HBT) in TCF pulp removes hexenuronic acids by 23% and reduces brightness reversion by 8.4%. Additional tests conducted to assess the effect of HexA on pulp refining revealed that these oxidizable structures introduce hydrophilicity in the pulp. Removing hexenuronic acids from TCF pulp alters its refining outcome as regards drainability ( degrees SR) and water retention capacity (%WRV); also, it leads to paper with comparable strength-related properties which requires no additional refining energy.